1. To identify the hydroxyapatite (HA) binding moiety of salivary agglutinin (SAG). 2. To functionalize and evaluate the protective property of the HA binding moiety against bacterial adherence and erosion.
Methods:
SAG consists of 13 Scavenger Receptor Cysteine-Rich (SRCR) domains, which are potentially involved in HA binding. Seven peptide fragments that constitute the amino acid sequence of a single SRCR domain of SAG were synthesized using Fmoc chemistry. The peptides were tested for their binding to the HA granules and the amount of peptide adsorbed to HA were quantified using Capillary Zone Electrophoresis. The HA binding peptide was conjugated to polyethylene glycol (PEG) to enhance its bacterial repellant property. The peptide and its PEGylated variant were evaluated for their anti-bacterial adherence property against Streptococcus mutans. The bacterial adherence to peptide coated wells was quantified with SYTO 9, a fluorescent DNA-binding probe. The anti-erosive property of the best HA binding peptide was evaluated by measuring the loss in weight of HA discs after in vitroexposure to citrate buffer. The HA discs were treated with peptide and human saliva before exposure to 0.1M citrate buffer, pH 3.0 for 30 min.
Results:
Of the seven peptides tested only one peptide designated as PC showed binding to the HA granules, suggesting that it is involved in binding of SAG to HA. In the bacterial adherence experiment, PC inhibited bacterial adherence by 20% while its PEGylated variant showed a 40% inhibition in bacterial adherence when compared with the control. Pretreatment of HA disc with PC followed by treatment with saliva showed a tendency towards protection against erosion when compared with protection by saliva pellicle alone.
Conclusions:
PC encompasses the HA binding domain of salivary agglutinin. PC and its PEGylated variant inhibit bacterial adherence. Furthermore, PC may also show anti-erosive property.